In vitro activity of ceftazidime-avibactam and comparators against Enterobacterales and Pseudomonas aeruginosa isolates from Central Europe and Israel, 2014-2017 and 2018.

Between 2014 and 2017, 6,662 Enterobacterales and 1,953 P. aeruginosa isolates were collected by 19 centers in four central European countries and Israel. A further 2,585 Enterobacterales and 707 P. aeruginosa isolates were collected in 2018 by 28 centers in seven European countries and Israel as part of the Antimicrobial Testing Leadership and Surveillance (ATLAS) study. A central laboratory performed antimicrobial susceptibility testing using broth microdilution panels according to Clinical Laboratory Standards Institute (CLSI) guidelines. Susceptibility rates among Enterobacterales were highest to ceftazidime-avibactam (≥98.5%), colistin (≥97.3%), and meropenem (≥95.8%). Ceftazidime-resistant and multidrug-resistant (MDR) Enterobacterales subsets were highly susceptible to ceftazidime-avibactam (≥94.9%) and colistin (≥94.7%). Susceptibility rates to colistin among all P. aeruginosa were ≥97.4% and were ≥96.3% among ceftazidime-resistant and MDR subsets. Susceptibility rates to ceftazidime-avibactam were 91.9% (2014-2017), 86.3% (2018) and, in common with comparator agents, were lower among ceftazidime-resistant (≥51.7%) and MDR isolates (≥57.1%).

C-tail mediated modulation of somatostatin receptor type-4 homo- and heterodimerizations and signaling.

Somatostatin receptors show great diversity in response to agonist mediated receptor-specific homo- and heterodimerizations. Here, using photobleaching-fluorescence resonance energy transfer, immunocytochemistry, western blot and co-immunoprecipitation, we investigated dimerization, trafficking, coupling to adenylyl cyclase and signaling of human somatostatin receptor-4 (hSSTR4) in HEK-293 cells. We also determined the role of the C-tail of hSSTR4 on physiological responses of the cells. wt-hSSTR4 exogenously expressed in HEK-293 cells exhibits constitutive dimerization, inhibits forskolin-stimulated cAMP, and displays agonist dependent changes in pERK1/2 and pERK5 expressions. Upon C-tail deletion, the receptor loses membrane expression and ability to dimerize and inhibition of cAMP and pERK5 however, displays several-fold increases in the expression of pERK1/2. Chimeric hSSTR4 with the C-tail of hSSTR5 functions like wt-hSSTR4, in contrast, with the C-tail of hSSTR1 functions like C-tail deleted hSSTR4. hSSTR4 dimerization and signaling are associated with increased cyclin-dependent-kinase p27(kip1) expression and inhibition of the cell proliferation. We also report heterodimerization between hSSTR4/hSSTR5, but not between hSSTR4/hSSTR1, with significant changes in receptor functions. Taken together, these data define a novel mechanism for the role of hSSTR4 in cell proliferation and modulation of signaling pathways.

Expression of somatostatin and somatostatin receptor subtypes in Apolipoprotein D (ApoD) knockout mouse brain: An immunohistochemical analysis.

Apolipoprotein D (ApoD) is widely distributed in central and peripheral nervous system. ApoD expression has been shown to increase in several neurodegenerative and neuropsychiatric disorders, as well as during regeneration in the nervous system. Like ApoD, in the central nervous system somatostatin (SST) is widely present and functions as neurotransmitter and neuromodulator. The biological effects of SST are mediated via binding to five high-affinity G-protein coupled receptors termed SSTR1-5. Mice lacking ApoD exhibit reduced SST labeling in cortex and hippocampus and increased expression in striatum and amygdala without any noticeable changes in substantia nigra. Changes in SSTRs expressions have been described in several neurodegenerative disorders including Alzheimer's, Parkinson's and Huntington's diseases. In the present study, using SSTR1-5 receptor-specific antibodies, we mapped their distribution in wild type (wt) and ApoD knockout (ApoD(-/-)) mouse brain. SSTR1-5 expression was observed both as membrane and cytoplasmic protein and display regions and receptor specific differences between wt and ApoD(-/-) mice brains. In cortex and hippocampus, SSTR subtypes like immunoreactivity are decreased in ApoD(-/-) mice brain. Unlike cortex and hippocampus, in the striatum of ApoD(-/-) mice, projection neurons showed increased SSTR immunoreactivity, as compared to wt. Higher SSTR subtypes immunoreactivity is seen in substantia nigra pars compacta (SNpc) whereas lower in substantia nigra pars reticulata (SNpr) of ApoD(-/-) mice brains as compared to wt. Whereas, amygdala displayed SSTR subtypes changes in different nuclei of ApoD(-/-) mice in comparison to wt mice brain. Taken together, our results describe receptor and region specific changes in SST and SSTR subtypes expression in ApoD(-/-) mice brain, which may be linked to specific neurological disorders.

Osmium tetroxide, 2,2'-bipyridine: electroactive marker for probing accessibility of tryptophan residues in proteins.

A complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) has been applied as a chemical probe of DNA structure as well as an electroactive DNA label. The Os,bipy has been known to form covalent adducts with pyrimidine DNA bases. Besides the pyrimidines, electrochemically active covalent adducts with Os,bipy are formed also by tryptophan (W) residues in peptides and proteins. In this paper we show that Os,bipy-treated proteins possessing W residues (such as avidin, streptavidin, or lysozyme) yield at the pyrolytic graphite electrode (PGE) a specific signal (peak alphaW) the potential of which differs from the potentials of signals produced by free Os,bipy or by Os,bipy-modified DNA. No such signal is observed with proteins lacking W (such as ribonuclease A or alpha-synuclein). Subpicomole amounts of W-containing proteins modified with Os,bipy can easily be detected using adsorptive transfer stripping voltammetry with the PGE. Binding of biotin to avidin interferes with Os,bipy modification of the protein, in agreement with the location of W residues within the biotin-binding site of avidin. These Ws are accessible for modification in the absence of biotin but hidden (protected from modification) in the avidin-biotin complex. The Os,bipy-modified avidin is unable to bind biotin, and its quarternary structure is disrupted. Analogous effects were observed with another biotin-binding protein, streptavidin. Our results demonstrate that modification of proteins with Os,bipy under conditions close to physiological, followed by a simple electrochemical analysis, can be applied in the microanalysis of protein structure and interactions.

Multiply osmium-labeled reporter probes for electrochemical DNA hybridization assays: detection of trinucleotide repeats.

In electrochemical DNA hybridization assays target or probe DNAs end-labeled with electroactive compounds have been frequently used. We show that multiple osmium labels yielding faradaic (at carbon or mercury electrodes) and catalytic signals (at mercury electrodes) can be easily covalently bound to DNA molecules. We use (GAA)(7) (T)(n) oligodeoxynucleotides (ODNs) with n ranging between 5 and 50. (T)(n) tails are selectively modified with osmium tetroxide,2,2'-bipyridine leaving the (GAA)(7) repeat intact for the DNA hybridization. These ODNs are applied as reporter probes (RP's) in DNA hybridization double-surface (DS) assay using magnetic beads for the DNA hybridization and pyrolytic graphite (PGE) or hanging mercury drop (HMDE) electrodes for the electrochemical detection. We show that in difference to the usual single-surface methods (where the RP has to be bound to target DNA near to the surface to communicate with the electrode) in the DS assay the RP can be bound to DNA regardless of its position and can used for the determination of the length of DNA repetitive sequences. Several fmols or about a hundred of amol of a RP with osmium-labeled (T)(50) tail can be detected at PGE and HMDE, respectively, at 1-2 min accumulation time.

Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize.

The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.

Square-wave voltammetric determination of cefoperazone in a bacterial culture, pharmaceutical drug, milk, and urine.

Use of square-wave voltammetry (SWV) for determination of cefoperazone (CFPZ) in some buffers, bacterial culture, urine, and milk is described. CFPZ provides a specific voltammetric signal which is affected by pH and solution components. Determination of CFPZ in Britton-Robinson buffer, pH 4.4, is sensitive with a low detection limit (about 0.5 nmol L(-1)). In a more complex medium (bacterial 2YT medium, pH 7.2) the detection limit was approximately 1.5 micromol L(-1). We provide evidence that SWV is a suitable and quick method for CFPZ determination in a culture of living bacteria without separation of biomass. We have found big differences between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in cultivation in the presence of CFPZ, depending on time. When CFPZ is cleaved by penicillinase, a new SWV peak b appears at more positive potentials. This peak rises both with increasing concentration of enzyme and with cleavage time while the original CFPZ peak is simultaneously decreasing. We determined the concentration of CFPZ in the drug Pathozone by the standard addition method and achieved good agreement with the declared value of CFPZ in the drug. With a simple pretreatment procedure it is possible to determine CFPZ in milk; for urine no pretreatment was required. Using SWV we could detect CFPZ concentrations as low as 500 nmol L(-1) in bovine milk and human urine.

Role of tumor suppressor p53 domains in selective binding to supercoiled DNA.

We showed previously that bacterially expressed full-length human wild-type p53b(1-393) binds selectively to supercoiled (sc)DNA in sc/linear DNA competition experiments, a process we termed supercoil-selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C-terminal domain (amino acids 347-382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361-393) protein (lacking the p53 tetramerization domain, amino acids 325-356) did not exhibit SCS binding while both dimeric mutant p53(319- 393)L344A and fusion protein GCN4-p53(347-393) were effective in SCS binding. Supershifting of p53(320-393)-scDNA complexes with monoclonal antibodies revealed that the amino acid region 375-378, constituting the epitope of the Bp53-10.1 antibody, plays a role in binding of the p53(320-393) protein to scDNA. Using electron microscopy we observed p53-scDNA nucleoprotein filaments produced by all the C-terminal proteins that displayed SCS binding in the gel electrophoresis experiments; no filaments formed with the monomeric p53(361- 393) protein. We propose a model according to which two DNA duplexes are compacted into p53-scDNA filaments and discuss a role for filament formation in recombination.

Differential pulse adsorptive stripping voltammetry of osmium-modified peptides.

Complexes of osmium tetroxide with nitrogen ligands were developed and used in our laboratory as probes of the DNA structure. Here, we show that the complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) can be used for modification and electrochemical detection of proteins at neutral pH. Salmon luteinizing hormone (SLH) containing two tryptophan (Trp) residues and human luteinizing hormone (HLH) containing one Trp were modified by Os,bipy and measured by differential pulse adsorptive stripping voltammetry (DPAdSV) at a hanging mercury drop electrode (HMDE). The intensity of the DPAdSV catalytic signals corresponded to the number of Trp residues in the peptide molecule. Decreasing pH of the background electrolyte from 6.6 to 3.8 led to the increase of DPAdSV signals, suggesting that at pH 3.8, the DPAdSV detection limit might be well below 1 ng/ml. Our results suggest that Os,bipy is potentially useful for chemical modification of proteins.

Voltammetric microanalysis of DNA adducts with osmium tetroxide,2,2'-bipyridine using a pyrolytic graphite electrode.

DNA and synthetic polynucleotides modified with a complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) produce specific voltammetric signals at pyrolytic graphite electrodes. Based on a sufficient potential separation between the peaks of Os,bipy-modified DNA (DNA-Os,bipy) and of free Os,bipy, and using an adsorptive transfer stripping voltammetric procedure involving extraction of free Os,bipy from the electrode by chloroform, DNA-Os,bipy can be determined in an excess of the free reagent. Under certain conditions, 140 pg of DNA-Os,bipy can be detected after a 5 min accumulation period. This analysis displays a more favorable sensitivity and a better selectivity for DNA structure than oxidation of DNA guanine moieties, and offers detection of osmium DNA markers at carbon electrodes.